• 1 Mendoza N, Li A, Gill A, et al. Filariasis: diagnosis and treatment. Dermatol Ther 2009; 22:475490.

  • 2 Genchi C, Rinaldi L, Mortarino M, et al. Climate and Dirofilaria infection in Europe. Vet Parasitol 2009; 163:286292.

  • 3 Simon F, Morchon R, Gonzalez-Miguel J, et al. What is new about animal and human dirofilariosis? Trends Parasitol 2009; 25:404409.

  • 4 Sironi M, Bandi C, Sacchi L, et al. Molecular evidence for a close relative of the arthropod endosymbiont Wolbachia in a filarial worm. Mol Biochem Parasitol 1995; 74:223227.

    • Search Google Scholar
    • Export Citation
  • 5 Bandi C, McCall JW, Genchi C, et al. Effects of tetracycline on the filarial worms B rugia pahangi and Dirofilaria immitis and their bacterial endosymbionts Wolbachia. Int J Parasitol 1999; 29:357364.

    • Search Google Scholar
    • Export Citation
  • 6 Taylor MJ, Bandi C, Hoerauf A. Wolbachia bacterial endosymbionts of filarial nematodes. Adv Parasitol 2005; 60:245284.

  • 7 Kramer LH, Tamarozzi F, Morchon R, et al. Immune response to and tissue localization of the Wolbachia surface protein (WSP) in dogs with natural heartworm (Dirofilaria immitis) infection. Vet Immunol Immunopathol 2005; 106:303308.

    • Search Google Scholar
    • Export Citation
  • 8 Kramer L, Simon F, Tamarozzi F, et al. Is Wolbachia complicating the pathological effects of Dirofilaria immitis infections? Vet Parasitol 2005; 133:133136.

    • Search Google Scholar
    • Export Citation
  • 9 McCall JW, Genchi C, Kramer L, et al. Heartworm and Wolbachia: therapeutic implications. Vet Parasitol 2008; 158:204214.

  • 10 Crowder CD, Matthews HE, Schutzer S, et al. Genotypic variation and mixtures of Lyme Borrelia in Ixodes ticks from North America and Europe. PLoS ONE 2010; 5:e10650.

    • Search Google Scholar
    • Export Citation
  • 11 Eshoo MW, Crowder CD, Li H, et al. Detection and identification of Ehrlichia species in blood by use of PCR and electrospray ionization mass spectrometry. J Clin Microbiol 2010; 48:472478.

    • Search Google Scholar
    • Export Citation
  • 12 Grant-Klein RJ, Baldwin CD, Turell MJ, et al. Rapid identification of vector-borne flaviviruses by mass spectrometry. Mol Cell Probes 2010; 24:219228.

    • Search Google Scholar
    • Export Citation
  • 13 Crowder CD, Rounds MA, Phillipson CA, et al. Extraction of total nucleic acids from ticks for the detection of bacterial and viral pathogens. J Med Entomol 2010; 47:8994.

    • Search Google Scholar
    • Export Citation
  • 14 Eshoo MW, Whitehouse CA, Zoll ST, et al. Direct broad-range detection of alphaviruses in mosquito extracts. Virology 2007; 368:286295.

    • Search Google Scholar
    • Export Citation
  • 15 Ecker JA, Massire C, Hall TA, et al. Identification of Acinetobacter species and genotyping of Acinetobacter baumannii by multilocus PCR and mass spectrometry. J Clin Microbiol 2006; 44:29212932.

    • Search Google Scholar
    • Export Citation
  • 16 Muddiman DC, Anderson GA, Hofstadler SA, et al. Length and base composition of PCR-amplified nucleic acids using mass measurements from electrospray ionization mass spectrometry. Anal Chem 1997; 69:15431549.

    • Search Google Scholar
    • Export Citation
  • 17 Duncan AW, Correa MT, Levine JF, et al. The dog as a sentinel for human infection: prevalence of Borrelia burgdorferi C6 antibodies in dogs from southeastern and mid-Atlantic states. Vector Borne Zoonotic Dis 2005; 5:101109.

    • Search Google Scholar
    • Export Citation
  • 18 Hamer SA, Tsao JI, Walker ED, et al. Use of tick surveys and serosurveys to evaluate pet dogs as a sentinel species for emerging Lyme disease. Am J Vet Res 2009; 70:4956.

    • Search Google Scholar
    • Export Citation
  • 19 Nelson CT, McCall JW, Rubin SB, et al. 2005 guidelines for the diagnosis, prevention and management of heartworm ( Dirofilaria immitis) infection in dogs. Vet Parasitol 2005; 133:255266.

    • Search Google Scholar
    • Export Citation

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Detection of heartworm infection in dogs via PCR amplification and electrospray ionization mass spectrometry of nucleic acid extracts from whole blood samples

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  • 1 Ibis Biosciences Incorporated, 1896 Rutherford Rd, Carlsbad, CA 92008.
  • | 2 Ibis Biosciences Incorporated, 1896 Rutherford Rd, Carlsbad, CA 92008.
  • | 3 Ibis Biosciences Incorporated, 1896 Rutherford Rd, Carlsbad, CA 92008.
  • | 4 Ibis Biosciences Incorporated, 1896 Rutherford Rd, Carlsbad, CA 92008.
  • | 5 Department of Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, NJ 07103.
  • | 6 Ibis Biosciences Incorporated, 1896 Rutherford Rd, Carlsbad, CA 92008.
  • | 7 Ibis Biosciences Incorporated, 1896 Rutherford Rd, Carlsbad, CA 92008.
  • | 8 Ibis Biosciences Incorporated, 1896 Rutherford Rd, Carlsbad, CA 92008.
  • | 9 Ibis Biosciences Incorporated, 1896 Rutherford Rd, Carlsbad, CA 92008.

Abstract

Objective—To develop and evaluate a rapid and accurate assay involving PCR amplification and electrospray ionization mass spectrometry of nucleic acid extracts from whole blood samples for the detection of Dirofilaria immitis infection in dogs.

Sample—Whole blood nucleic acid extracts from 29 dogs experimentally infected with D immitis (and in which circulating D immitis antigen was detected) and 10 uninfected dogs.

Procedures—16 of the 29 whole blood samples from infected dogs were examined at the time of collection for circulating microfilaria. Nucleic acids were extracted from all whole blood specimens and underwent PCR amplification with 12 PCR primer pairs designed to detect a wide range of pathogens (including the Wolbachia endosymbiont of D immitis) and electrospray ionization mass spectrometry.

Results—On the basis of assay results, heartworm infection was detected in 13 of 13 antigen-positive dogs of unknown microfilaria status, 11 of 11 antigen-positive dogs with circulating microfilaria, 0 of 3 antigen-positive dogs tested at 3 months after larval infection, 0 of 2 antigen-positive dogs with occult infections, and 0 of 10 uninfected dogs.

Conclusions and Clinical Relevance—With the assay under investigation, it was possible to identify D immitis infection in dogs with circulating microfilaria via detection of the obligate Wolbachia endosymbiont of D immitis. It was not possible to identify dogs with occult infections, which suggested that circulating microfilaria must be present to detect infection with this assay, although further studies would be required to verify that finding.

Contributor Notes

Supported by the National Institute of Allergy and Infectious Diseases grant No. 2R44AI077156-02.

The content of this report is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases or the National Institutes of Health.

Address correspondence to Dr. Crowder (christopher.crowder@abbott.com).